Recent studies suggest that tissue factor (TF) plays an important role in biological processes other than blood coagulation, including inflammation, angiogenesis and tumor metastasis. How does TF, whose principal role is to support clotting factor Vila (Vila) in initiating the coagulation cascade, affect various pathophysiological processes? Recent studies from our laboratory and others show that Vila binding to TF not only initiates clotting but also induces cell signaling, independent of down-stream coagulation proteases, by activating G-protein-coupled protease activated receptors (PARs). Our studies show that Vila binding to TF on tumor cells induces matricellular proteins and chemokines via PAR2-mediated cell signaling. Our preliminary data of heterologous desensitization and analysis of PAR2 cleavage indicate that only a small fraction of PAR2 may be activated by TF-Vlla at the cell surface, yet TF-Vlla can induce robust cell signaling via PAR2. The central goal of this application is to understand how TF-Vlla induces the cell signaling efficiently and whether TF-VIla-induced cell signaling, as indicated in in vitro studies, contributes to tumor metastasis. In Aim 1, we will determine TF-Vlla and TF-Vlla-Xa cleavage of PAR2 and PAR1, and how this relates to TF-VIla/TF-Vlla-Xa-induced cell signaling in tumor cells. In Aim 2, we will test a novel hypothesis that compartmentalization of TF, PAR2 and the effector molecules in plasma membrane microdomains facilitates efficient TF-Vlla-induced cell signaling. Finally, in Aim 3, we will determine the role of TFVI la/PAR2-mediated cell signaling in tumor metastasis. We will utilize the state-of-the-art techniques, such as confocal and electron microscopy, and gene silencing to achieve the stated goals. Data from these studies will answer many important questions concerning the mode of TF-Vlla-induced cell signaling and its importance in pathophysiology. Relevance to public health: Tissue factor expression in cancer cells is responsible for the hypercoagulable state presented in many cancers, and contributes to the pathogenesis of cancer progression and tumor metastasis. The proposed studies will provide valuable information in understanding how TF contributes to tumorigenesis. These studies will also provide clues on potential therapeutic targets for prevention of cancer progression.